Proteomic analysis across patient iPSC-based models and human post-mortem hippocampal tissue reveals early cellular dysfunction and progression of Alzheimer's disease pathogenesis.

The hippocampus is a primary region affected in Alzheimer's disease (AD). Because AD postmortem brain tissue is not available prior to symptomatic stage, we lack understanding of early cellular pathogenic mechanisms. To address this issue, we examined the cellular origin and progression of AD pathogenesis by comparing patient-based model systems including iPSC-derived brain cells transplanted into the mouse brain hippocampus. Proteomic analysis of the graft enabled the identification of pathways and network dysfunction in AD patient brain cells, associated with increased levels of Aß-42 and ß-sheet structures. Interestingly, the host cells surrounding the AD graft also presented alterations in cellular biological pathways. Furthermore, proteomic analysis across human iPSC-based models and human post-mortem hippocampal tissue projected coherent longitudinal cellular changes indicative of early to end stage AD cellular pathogenesis. Our data showcase patient-based models to study the cell autonomous origin and progression of AD pathogenesis.

Apolipoprotein E intersects with amyloid-ß within neurons.

Apolipoprotein E4 (ApoE4) is the most important genetic risk factor for Alzheimer's disease (AD). Among the earliest changes in AD is endosomal enlargement in neurons, which was reported as enhanced in ApoE4 carriers. ApoE is thought to be internalized into endosomes of neurons, whereas ß-amyloid (Aß) accumulates within neuronal endosomes early in AD. However, it remains unknown whether ApoE and Aß intersect intracellularly. We show that internalized astrocytic ApoE localizes mostly to lysosomes in neuroblastoma cells and astrocytes, whereas in neurons, it preferentially localizes to endosomes-autophagosomes of neurites. In AD transgenic neurons, astrocyte-derived ApoE intersects intracellularly with amyloid precursor protein/Aß. Moreover, ApoE4 increases the levels of endogenous and internalized Aß42 in neurons. Taken together, we demonstrate differential localization of ApoE in neurons, astrocytes, and neuron-like cells, and show that internalized ApoE intersects with amyloid precursor protein/Aß in neurons, which may be of considerable relevance to AD.

Cerebral Aß deposition precedes reduced cerebrospinal fluid and serum Aß42/Aß40 ratios in the AppNL-F/NL-F knock-in mouse model of Alzheimer's disease.

BACKGROUND: Aß42/Aß40 ratios in cerebrospinal fluid (CSF) and blood are reduced in preclinical Alzheimer's disease (AD), but their temporal and correlative relationship with cerebral Aß pathology at this early disease stage is not well understood. In the present study, we aim to investigate such relationships using App knock-in mouse models of preclinical AD. METHODS: CSF, serum, and brain tissue were collected from 3- to 18-month-old AppNL-F/NL-F knock-in mice (n = 48) and 2-18-month-old AppNL/NL knock-in mice (n = 35). The concentrations of Aß42 and Aß40 in CSF and serum were measured using Single molecule array (Simoa) immunoassays. Cerebral Aß plaque burden was assessed in brain tissue sections by immunohistochemistry and thioflavin S staining. Furthermore, the concentrations of Aß42 in soluble and insoluble fractions prepared from cortical tissue homogenates were measured using an electrochemiluminescence immunoassay. RESULTS: In AppNL-F/NL-F knock-in mice, Aß42/Aß40 ratios in CSF and serum were significantly reduced from 12 and 16 months of age, respectively. The initial reduction of these biomarkers coincided with cerebral Aß pathology, in which a more widespread Aß plaque burden and increased levels of Aß42 in the brain were observed from approximately 12 months of age. Accordingly, in the whole study population, Aß42/Aß40 ratios in CSF and serum showed a negative hyperbolic association with cerebral Aß plaque burden as well as the levels of both soluble and insoluble Aß42 in the brain. These associations tended to be stronger for the measures in CSF compared with serum. In contrast, no alterations in the investigated fluid biomarkers or apparent cerebral Aß plaque pathology were found in AppNL/NL knock-in mice during the observation time. CONCLUSIONS: Our findings suggest a temporal sequence of events in AppNL-F/NL-F knock-in mice, in which initial deposition of Aß aggregates in the brain is followed by a decline of the Aß42/Aß40 ratio in CSF and serum once the cerebral Aß pathology becomes significant. Our results also indicate that the investigated biomarkers were somewhat more strongly associated with measures of cerebral Aß pathology when assessed in CSF compared with serum.

APOE in the bullseye of neurodegenerative diseases: impact of the APOE genotype in Alzheimer's disease pathology and brain diseases.

ApoE is the major lipid and cholesterol carrier in the CNS. There are three major human polymorphisms, apoE2, apoE3, and apoE4, and the genetic expression of APOE4 is one of the most influential risk factors for the development of late-onset Alzheimer's disease (AD). Neuroinflammation has become the third hallmark of AD, together with Amyloid-ß plaques and neurofibrillary tangles of hyperphosphorylated aggregated tau protein. This review aims to broadly and extensively describe the differential aspects concerning apoE. Starting from the evolution of apoE to how APOE's single-nucleotide polymorphisms affect its structure, function, and involvement during health and disease. This review reflects on how APOE's polymorphisms impact critical aspects of AD pathology, such as the neuroinflammatory response, particularly the effect of APOE on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-ß load, tau pathology, autophagy, and cell-cell communication. We discuss influential factors affecting AD pathology combined with the APOE genotype, such as sex, age, diet, physical exercise, current therapies and clinical trials in the AD field. The impact of the APOE genotype in other neurodegenerative diseases characterized by overt inflammation, e.g., alpha- synucleinopathies and Parkinson's disease, traumatic brain injury, stroke, amyotrophic lateral sclerosis, and multiple sclerosis, is also addressed. Therefore, this review gathers the most relevant findings related to the APOE genotype up to date and its implications on AD and CNS pathologies to provide a deeper understanding of the knowledge in the APOE field.

Aß/Amyloid Precursor Protein-Induced Hyperexcitability and Dysregulation of Homeostatic Synaptic Plasticity in Neuron Models of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly seen as a disease of synapses and diverse evidence has implicated the amyloid-ß peptide (Aß) in synapse damage. The molecular and cellular mechanism(s) by which Aß and/or its precursor protein, the amyloid precursor protein (APP) can affect synapses remains unclear. Interestingly, early hyperexcitability has been described in human AD and mouse models of AD, which precedes later hypoactivity. Here we show that neurons in culture with either elevated levels of Aß or with human APP mutated to prevent Aß generation can both induce hyperactivity as detected by elevated calcium transient frequency and amplitude. Since homeostatic synaptic plasticity (HSP) mechanisms normally maintain a setpoint of activity, we examined whether HSP was altered in AD transgenic neurons. Using methods known to induce HSP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and that AD transgenic neurons have an impaired adaptation of calcium transients to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we show that both APP and Aß influence neuronal activity and that mechanisms of HSP are disrupted in primary neuron models of AD.

Sphingosine 1-Phoshpate Receptors are Located in Synapses and Control Spontaneous Activity of Mouse Neurons in Culture.

Sphingosine-1-phosphate (S1P) is best known for its roles as vascular and immune regulator. Besides, it is also present in the central nervous system (CNS) where it can act as neuromodulator via five S1P receptors (S1PRs), and thus control neurotransmitter release. The distribution of S1PRs in the active zone and postsynaptic density of CNS synapses remains unknown. In the current study, we investigated the localization of S1PR1-5 in synapses of the mouse cortex. Cortical nerve terminals purified in a sucrose gradient were endowed with all five S1PRs. Further subcellular fractionation of cortical nerve terminals revealed S1PR2 and S1PR4 immunoreactivity in the active zone of presynaptic nerve terminals. Interestingly, only S1PR2 and S1PR3 immunoreactivity was found in the postsynaptic density. All receptors were present outside the active zone of nerve terminals. Neurons in the mouse cortex and primary neurons in culture showed immunoreactivity against all five S1PRs, and Ca2+ imaging revealed that S1P inhibits spontaneous neuronal activity in a dose-dependent fashion. When testing selective agonists for each of the receptors, we found that only S1PR1, S1PR2 and S1PR4 control spontaneous neuronal activity. We conclude that S1PR2 and S1PR4 are located in the active zone of nerve terminals and inhibit neuronal activity. Future studies need to test whether these receptors modulate stimulation-induced neurotransmitter release.

Correlative imaging to resolve molecular structures in individual cells: Substrate validation study for super-resolution infrared microspectroscopy.

Light microscopy has been a favorite tool of biological studies for almost a century, recently producing detailed images with exquisite molecular specificity achieving spatial resolution at nanoscale. However, light microscopy is insufficient to provide chemical information as a standalone technique. An increasing amount of evidence demonstrates that optical photothermal infrared microspectroscopy (O-PTIR) is a valuable imaging tool that can extract chemical information to locate molecular structures at submicron resolution. To further investigate the applicability of sub-micron infrared microspectroscopy for biomedical applications, we analyzed the contribution of substrate chemistry to the infrared spectra acquired from individual neurons grown on various imaging substrates. To provide an example of correlative immunofluorescence/O-PTIR imaging, we used immunofluorescence to locate specific organelles for O-PTIR measurement, thus capturing molecular structures at the sub-cellular level directly in cells, which is not possible using traditional infrared microspectroscopy or immunofluorescence microscopy alone.

Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

Monitoring the interactions between alpha-synuclein and Tau in vitro and in vivo using bimolecular fluorescence complementation.

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-ß (Aß) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

Astrocytic and Neuronal Apolipoprotein E Isoforms Differentially Affect Neuronal Excitability.

Synaptic changes and neuronal network dysfunction are among the earliest changes in Alzheimer's disease (AD). Apolipoprotein E4 (ApoE4), the major genetic risk factor in AD, has been shown to be present at synapses and to induce hyperexcitability in mouse knock-in brain regions vulnerable to AD. ApoE in the brain is mainly generated by astrocytes, however, neurons can also produce ApoE under stress conditions such as aging. The potential synaptic function(s) of ApoE and whether the cellular source of ApoE might affect neuronal excitability remain poorly understood. Therefore, the aim of this study was to elucidate the synaptic localization and effects on neuronal activity of the two main human ApoE isoforms from different cellular sources in control and AD-like in vitro cultured neuron models. In this study ApoE is seen to localize at or near to synaptic terminals. Additionally, we detected a cellular source-specific effect of ApoE isoforms on neuronal activity measured by live cell Ca2+ imaging. Neuronal activity increases after acute but not long-term administration of ApoE4 astrocyte medium. In contrast, ApoE expressed by neurons appears to induce the highest neuronal firing rate in the presence of ApoE3, rather than ApoE4. Moreover, increased neuronal activity in APP/PS1 AD transgenic compared to wild-type neurons is seen in the absence of astrocytic ApoE and the presence of astrocytic ApoE4, but not ApoE3. In summary, ApoE can target synapses and differentially induce changes in neuronal activity depending on whether ApoE is produced by astrocytes or neurons. Astrocytic ApoE induces the strongest neuronal firing with ApoE4, while the most active and efficient neuronal activity induced by neuronal ApoE is caused by ApoE3. ApoE isoforms also differentially affect neuronal activity in AD transgenic compared to wild-type neurons.

APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Correlative optical photothermal infrared and X-ray fluorescence for chemical imaging of trace elements and relevant molecular structures directly in neurons.

Alzheimer's disease (AD) is the most common cause of dementia, costing about 1% of the global economy. Failures of clinical trials targeting amyloid-ß protein (Aß), a key trigger of AD, have been explained by drug inefficiency regardless of the mechanisms of amyloid neurotoxicity, which are very difficult to address by available technologies. Here, we combine two imaging modalities that stand at opposite ends of the electromagnetic spectrum, and therefore, can be used as complementary tools to assess structural and chemical information directly in a single neuron. Combining label-free super-resolution microspectroscopy for sub-cellular imaging based on novel optical photothermal infrared (O-PTIR) and synchrotron-based X-ray fluorescence (S-XRF) nano-imaging techniques, we capture elemental distribution and fibrillary forms of amyloid-ß proteins in the same neurons at an unprecedented resolution. Our results reveal that in primary AD-like neurons, iron clusters co-localize with elevated amyloid ß-sheet structures and oxidized lipids. Overall, our O-PTIR/S-XRF results motivate using high-resolution multimodal microspectroscopic approaches to understand the role of molecular structures and trace elements within a single neuronal cell.

Neuronal spreading and plaque induction of intracellular Aß and its disruption of Aß homeostasis.

The amyloid-beta peptide (Aß) is thought to have prion-like properties promoting its spread throughout the brain in Alzheimer's disease (AD). However, the cellular mechanism(s) of this spread remains unclear. Here, we show an important role of intracellular Aß in its prion-like spread. We demonstrate that an intracellular source of Aß can induce amyloid plaques in vivo via hippocampal injection. We show that hippocampal injection of mouse AD brain homogenate not only induces plaques, but also damages interneurons and affects intracellular Aß levels in synaptically connected brain areas, paralleling cellular changes seen in AD. Furthermore, in a primary neuron AD model, exposure of picomolar amounts of brain-derived Aß leads to an apparent redistribution of Aß from soma to processes and dystrophic neurites. We also observe that such neuritic dystrophies associate with plaque formation in AD-transgenic mice. Finally, using cellular models, we propose a mechanism for how intracellular accumulation of Aß disturbs homeostatic control of Aß levels and can contribute to the up to 10,000-fold increase of Aß in the AD brain. Our data indicate an essential role for intracellular prion-like Aß and its synaptic spread in the pathogenesis of AD.

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer's Disease.

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of ß-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of ß-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-ß, α-synuclein) and (amyloid-ß, Tau) neuron-like cells display changes in ß-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.

Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss.

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and ß-amyloid (Aß) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied neuronal aging using normal-aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identified the upregulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aß production in vitro and in vivo. The increased APP endocytosis may contribute to the early endosome enlargement observed in the aged brain. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aß production inhibition reverts synaptic decline in aged neurons, whereas Aß accumulation, promoted by endocytosis upregulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis upregulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease. This article has an associated First Person interview with the first author of the paper.

Accumulation of cellular prion protein within ß-amyloid oligomer plaques in aged human brains.

Alzheimer's disease (AD) is the main cause of dementia, and ß-amyloid (Aß) is a central factor in the initiation and progression of the disease. Different forms of Aß have been identified as monomers, oligomers, and amyloid fibrils. Many proteins have been implicated as putative receptors of respective forms of Aß. Distinct forms of Aß oligomers are considered to be neurotoxic species that trigger the pathophysiology of AD. It was reported that cellular prion protein (PrPC ) is one of the most selective and high-affinity binding partners of Aß oligomers. The interaction of Aß oligomers with PrPC is important to synaptic dysfunction and loss. The binding of Aß oligomers to PrPC has mostly been studied with synthetic peptides, cell culture, and murine models of AD by biochemical and biological methods. However, the molecular mechanisms underlying the relationship between Aß oligomers and PrPC remain unclear, especially in the human brain. We immunohistochemically investigated the relationship between Aß oligomers and PrPC in human brain tissue with and without amyloid pathology. We histologically demonstrate that PrPC accumulates with aging in human brain tissue even prior to AD mainly within diffuse-type amyloid plaques, which are composed of more soluble Aß oligomers without stacked ß-sheet fibril structures. Our results suggest that PrPC accumulating plaques are associated with more soluble Aß oligomers, and appear even prior to AD. The investigation of PrPC accumulating plaques may provide new insights into AD.